直肠癌新辅助放化疗后残余肿瘤侵袭转移相关生物学特性的变化及其机制

Neoadjuvant chemoradiotherapy is important among the multidisciplinary management of rectal cancer. Recent years it has been concerned that chemotherapy and radiotherapy can effect the biological behavior of tumor cells. In our previous study it was found that residual hepatocellular carcinoma cells after chemotherapy and radiation gained enhanced metastatic ability with relevance to epithelial mesenchymal transition. It is not clear whether the same phenomenon appears in the chemoradiotherapy of rectal cancer and what is the key gene responsible for it. More importantly, there is still not enough evidence from human rectal cancer tissue to prove the chemo and radiation related deterioration of biological behavior in tumor cells. This study is proposed to: 1) establish the model of in vitro chemoradiotherapy on rectal tumor cells , detect the changes of biological features concerning invasion and metastatic ability of residual colorectal cancer cells and determine the key gene responsible for it. 2) search and prove the changes of molecular phenotypes and the expression of the key gene in residual rectal cancer after chemoradiotherapy, analyze the relationship between the expression of the key gene and the prognosis of the patients. 3)explore the possibility of modulating the biological behavior of residual rectal cancer cells through blocking the expression or function of the key gene. This program will reveal the changes of biological behavior of residual rectal cancer cells after chemoradiotherapy and provide the scientific evidence to improve the outcome of multidisciplinary management of rectal cancer.

新辅助同期放化疗是直肠癌综合治疗的重要组成部分。近年来，放化疗导致残余肿瘤生物学行为变化的现象开始受到重视。我们前期研究发现，放化疗后的残余肝癌出现侵袭转移能力增强的现象，其原因与上皮-间质转化等机制有关。目前尚不清楚在直肠癌的放化疗过程中是否也存在类似现象，其关键的调控基因是什么。更为重要的是，目前尚未从人肿瘤组织中找到放化疗导致肿瘤细胞生物学行为恶化的有力证据。本项目拟通过①建立人结直肠癌细胞体外同期放化疗模型，研究放化疗后残余癌细胞侵袭转移相关生物学特性的变化趋势，利用基因芯片筛选与之相关的关键基因。② 在人直肠癌标本中寻找和验证放化疗后残余癌细胞的分子表型变化及关键基因的表达，分析其与患者预后的关系③探索阻断关键基因的表达和（或）功能，改善放化疗后残余直肠癌细胞生物学特性的可能。本项目将为揭示新辅助放化疗后残余直肠癌生物学特性的变化及其机制，提高直肠癌综合治疗疗效提供科学依据。

近年来，放化疗导致残余肿瘤细胞生物学行为变化的现象开始受到重视。当大多数研究把提高结直肠癌细胞放化疗疗效的重点放在如何减少肿瘤负荷时，放化疗对结直肠癌细胞侵袭转移生物学行为的影响尚不完全清楚。本研究主要通过细胞实验、动物实验以及对直肠癌患者的肿瘤组织标本的研究探索放化疗对结直肠癌细胞侵袭转移生物学行为的影响及其相关机制。细胞移动实验结果显示，与母细胞相比，放化疗后的残余HCT116 和HT29细胞移动能力均显著增强（15.33±5.07 vs 46.56 ±7.97, p<0.01 和15.83±9.88 vs 59.16±20.73, p<0.01）。侵袭实验结果显示了同样的趋势，放化疗后的残余HCT116 和HT29细胞侵袭能力均显著增强（59.17±13.34 vs 149.63±7.65, p<0.01 和35.97±2.89 vs 96.00±8.13, p<0.01）。细胞免疫荧光结果显示，放化疗后的残余肿瘤细胞呈现上皮-间质转化现象。放化疗后的残余肿瘤细胞在动物体内呈现出侵袭转移能力增强的趋势(0/6 vs 1/6)，但无统计学差异。生物芯片检测出导致放化疗后残余肿瘤细胞和母细胞生物学行为差异的mRNA和LncRNA表达谱，其中LINC00152为关键的分子靶点。运用siRNA干扰技术对放化疗后的HCT116残癌细胞进行LINC00152干扰，与未干扰组相比，干扰组残癌细胞的运动能力显著减弱(206.00±29.46 vs 65.00 ±24.06, p<0.01)，侵袭能力也显著减弱(57.00 ±12.53 vs 9.67 ±4.73, p<0.01)。对接受新辅助放化疗的直肠癌患者放化疗前、后的标本进行研究发现，与放化疗前的直肠癌组织相比，免疫组化结果提示放化疗后的直肠癌组织中肿瘤细胞发生上皮-间质转化现象, PCR实验结果提示放化疗后的直肠癌细胞中LINC00152表达显著增强。本研究结果显示，放化疗后残余肿瘤细胞呈现出运动、侵袭能力增强的现象，其原因与肿瘤细胞mRNA 和LncRNA调控机制有关，LINC00152是其中关键的分子靶点。放化疗后直肠癌细胞的这种生物学行为变化趋势可能同样存在于直肠癌患者的临床治疗实践中，对该现象的进一步深入研究可能为提高直肠癌患者放化疗的综合疗效开辟新的途径。